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Abstract

Highly exfoliated sulfonated graphene sheets (SGSs), an alternative to graphene oxide
and graphene derivatives, were synthesized, characterized, and applied to liver cancer
cells in vitro. Cytotoxicity profiles were obtained using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, WST-1[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, and lactate dehydrogenase release colorimetric assays. These particles
were found to be non-toxic across the concentration range of 0.1 to 10 μg/ml. Internalization
of SGSs was also studied by means of optical and electron microscopy. Although not
conclusive, high-resolution transmission and scanning electron microscopy revealed
variant internalization behaviors where some of the SGS became folded and compartmentalized
into tight bundles within cellular organelles. The ability for liver cancer cells
to internalize, fold, and compartmentalize graphene structures is a phenomenon not
previously documented for graphene cell biology and should be further investigated.

Keywords:

Cytotoxicity; Sulfonated graphene sheets; Cancer cells

Background

Carbon-derived nanoparticles (NPs) such as single- and multi-walled carbon nanotubes,
fullerenes, and graphene are all receiving attention because of their interesting
and unusual electronic
[1], thermal
[2], and mechanical
[3] properties. We have recently demonstrated a facile route towards the synthesis of
nanosized water-soluble sulfonated graphene sheets (SGSs) that use graphite as the
starting material
[4]. This method relies on the addition of phenyl radicals with subsequent sulfonation
of the phenyl groups and produces fewer defects and holes that can be introduced into
the graphene plates through the use of heavy sonication. A possible application of
these SGSs is within the medical sector due to their enhanced solubility (compared
to other graphene derivatives) and potential for surface modifications for attachment
of biomolecules and drugs. However, the interaction of SGSs with biological systems
has yet to be investigated and is the basis of the work described herein.

To date, much of the biological work regarding graphene has focused on assessing the
cytotoxicity, cell adhesion, proliferation, and antibacterial properties of graphene oxide (GO)
[5-8] as well as biodistribution, toxicology, and internalization of various suspensions
of GO complexes. These include 125I and 188Re radioisotope-labeled GO
[9,10], PEGylated GO for cellular imaging and delivery of water-insoluble cancer drugs
[11-13], and the imaging and treatment of brain, lung, and breast xenograft tumors in mice
through the use of photothermal light therapy from the absorption of near-infrared
(NIR) light by PEGylated GO with fluorescent Cy7 probes
[14].

Toxicity analysis (in vitro) of GO (prepared using chemical vapor deposition or the modified Hummers method
[15]) on lung
[16,17] and neuronal
[18] cell lines (A549 and PC12, respectively) has shown concentration-dependent cytotoxicity.
The exact mechanism of cell death from GO remains uncertain although a slight increase
in lactate dehydrogenase (LDH) from cells, generation of reactive oxygen species,
and weak activation of a caspase-3-mediated apoptosis pathway have all been reported.
These reports suggest GO cytotoxicity from either direct cellular membrane damage
or activation of natural cellular suicide mechanisms.

Similarly, in vivo mouse toxicology studies have shown that GO nanoplatelets of diameters 10 to 700
nm apparently cause no acute toxicities at low doses
[9,10]. However, at high doses (10 mg/kg), significant pathological changes such as granulomatous
lesions, pulmonary edema, inflammatory cell infiltration, and fibrosis were observed
throughout the lungs.

In light of the potential applications of graphene materials in drug delivery, imaging,
and thermal therapy, but with limitations due to cytotoxicity of GO, we sought to
investigate the in vitro interaction of our highly water-soluble SGS with liver cancer cells. Our initial
studies using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), WST-1[2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1), and LDH colorimetric assays have shown that SGSs are non-toxic
up to concentrations of 10 μg/ml. We also show that liver cancer cell lines (SNU449
and Hep3B) can internalize SGSs of diameters up to 5 μm, which in some cases are comparable
to the size of the cells themselves. Preliminary electron microscopy analysis also
suggests that these cells are capable of folding and compartmentalizing sheets of
smaller sizes (approximately 1.41 μm) although more work should be undertaken to validate.

Since graphene has been documented to be the hardest material known
[3], this unique behavior of water-soluble SGS with cells is counterintuitive and suggests
a novel finding that may have far-reaching applications in biology and medicine such
as enhanced drug delivery (due to the large graphene surface area), and should warrant
further investigation. Given that these SGSs are non-toxic up to 10 μg/ml, we feel
they can be used as an adequate scaffold to simultaneously attach targeting moieties
such as EGFR antibodies (e.g., cetuximab, C225) and chemo-agents such as doxorubicin
and gemcitabine in a bid to treat hepatocellular carcinoma legions. The use of a targeted
thermal ‘trigger’ such as photon activation (i.e., NIR light) or radiofrequency electric
fields could allow these SGSs to release their cargo into the cells upon irradiation
by a stimuli. Such a scheme has recently been reported using cisplatin-filled ultra-short
carbon nanotubes that release their cargo upon exposure to high-intensity radiofrequency
electric fields
[19].

Methods

Sample preparation and characterization

Samples were obtained from Mukherjee et al.
[4]. In their technique, highly exfoliated SGSs can be synthesized by sulfonation of
commercially available graphite (particle size < 20 μm) in oleum to overcome the cohesive
van deer Waals attractions between adjacent sheets. Their exfoliation method was selected
over the procedure by Si et al.
[20] as it produces fewer defects and holes that can be introduced into the graphene plates
through the use of heavy sonication. In brief, the addition of benzoyl peroxide to
a suspension of graphite in benzene at 75°C to 80°C provided phenylated graphite,
the sulfonation of which by oleum leads to highly-exfoliated graphene sheets which
can be further converted into a sodium salt by the addition of 1 M sodium hydroxide.
This material, in powder form, is highly soluble in water (approximately 2.1 mg/ml)
due to the p-sulphonated substituents, and it is relatively free of basal plane defects
that typically result from the removal of the oxygen functionality of comparable GO
compounds.

The SGSs in powder form were characterized via Raman spectroscopy, thermogravimetric
analysis (TGA), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy
(AFM). Raman spectra of the initial graphite material were compared to SGSs using
a Renishaw 1000 micro-Raman system (Gloucestershire, UK) with a 514-nm excitation
laser source. Multiple spectra were taken
[3-5] and normalized to the G band. TGA data were taken using a model SDT 2960 TA (TA Instruments,
Newcastle, DE, USA) instrument in both an argon and air atmosphere. Samples were first
degassed at 80°C and then heated at 10°C/min to 700°C and held there for 20 min. This
allowed for accurate percentage determination of the sodium sulfonate groups (approximately
6%). XPS data were obtained using a physical electronics (PHI QUENTERA, Chanhassen,
MN, USA) XPS/ESCA system with a base pressure of 5 × 10−9 Torr. A monochromatic Al X-ray source at 100 W was used with a pass energy of 26
eV and a 45° takeoff angle. The beam diameter was 100.0 μm. Low- and high-resolution
survey scans of the elements C, O, Na, and S were taken. At least two separate locations
were analyzed for each sample. For AFM studies, aqueous solution of SGSs at 50 mg/l
was drop-cast onto freshly cleaved mica and placed in a desiccator for 24 h prior
to imaging. Tapping-mode AFM images were taken in air under ambient conditions on
a Digital Instruments Nanoscope IIIA (Digital Instruments, Tonawanda, NY, USA).

Cell culture studies

SGS cytotoxicity was investigated using multiple assays. Cell membrane integrity was
evaluated using a LDH release assay. Cell proliferation/metabolic activity was investigated
using the popular MTT and WST-1 colorimetric assays. For in vitro experiments, approximately 3 mg of the SGS powder was added to 3 ml of phosphate-buffered
saline (PBS) to create two suspensions of concentration 1,000 μg/ml. All samples were
sterilized for 20 min using a bench-top UV sterilizer. SNU449 and Hep3B liver cancer
cells were utilized for the experiments (American Type Culture Collection, Bethesda,
MD, USA). The cells were maintained in standard culture conditions with 10% fetal
calf serum and penicillin/streptomycin at 37°C. Cell morphology was analyzed using
real-time bright-field optical imaging.

MTT assay

SNU449 and Hep3B cells were plated in 96-well plates at a density between 1,000 to
2,000 cells per well. After 24 h, the SNU449 and Hep3B cells were exposed to increasing
concentrations (0.1, 1.0, 10, and 100 μg/ml) of SGSs in PBS and were compared to a
PBS only control group (all suspensions were lightly sonicated for 5 min before use).
Cell viability was assessed at 24, 72, and 120 h after exposure to the SGSs. At each
time point, the media (100 μl) was carefully aspirated and replaced before adding
MTT reagent to each well and incubating for 4 h. The media was again carefully removed,
and purple formazan crystals were dissolved in dimethyl sulfoxide (DMSO). The 96-well
plates were then spun down at 3,500 rpm for 5 min (to force any cells/SGS debris to
the bottom of the well) where 50 μl of the colored media was withdrawn and placed
into a fresh 96-well plate. Absorbance was interpreted at 570 nm for each well using
a SPECTROstar Nano plate reader (BMG Labtech Inc., Cary, NC, USA).

WST-1 assay

These studies were prepared similar to the MTT assay but for a shorter duration (24,
48, and 72 h) as MTT assays showed that maximum toxicity occurred at 72 h. Also, it
was harder to keep the control cells from overgrowing for times greater than 72 h.
At each time point, WST-1 reagent was added to each well and incubated for 3 h. The
96-well plate was then spun down at 3,500 rpm for 5 min (to force any cells/SGS debris
to the bottom of the well) where 50 μl of the colored media was withdrawn and placed
into a fresh 96-well plate. This negated any effects from inherent SGS absorption
as all the SGSs were contained at the bottom of the discarded well. Absorbance was
interpreted at 450 nm for each well using a SPECTROstar Nano plate reader (BMG Labtech
Inc.).

LDH assay

SNU449 and HEP3B cells were exposed to various concentrations of SGSs (0.1, 1.0, 10.0,
and 100 μg/ml) for 24, 48, and 72 h, and the cell-free supernatant was removed. Maximum
LDH release was obtained by exposing the cells to a 2% Triton-X 100 solution to permeabilize
the membranes. LDH activity was determined by the use of a cytotoxicity detection
kit purchased from Roche Applied Science (Indianapolis, IN, USA). Aliquots of the
cell culture media from the SGS-exposed samples, untreated samples, and the permeabilized
samples were added to a 96-well plate, and an equal volume of LDH cytotoxicity detection
reagent was added. The 96-well plates were read on a spectrophotometer, and the absorbance
at 492 nm was measured. Calculations were performed as per the recommendations of
the kit. To show that SGS does not interfere with the kit, cells were permeabilized
with a 2% Triton-X 100 solution. The lysate was incubated with various concentrations
of SGS for 24 h. No difference was observed for any of the control samples indicating
that SGSs do not interfere with the assay.

Flow cytometry

Viability was measured with flow cytometry (LSRII, BD Biosciences, Franklin, NJ, USA)
as described previously
[21]. Briefly, cell media was aspirated, and the adherent cells were collected after trypsinization.
Each sample was washed and stained with annexin V-FITC and propidium iodide (PI) without
fixation or permeabilization. Annexin V is a protein that binds to phosphatidylserine,
which is externalized in apoptotic cells. Propidium iodide fluoresces when it is bound
to DNA in membrane-damaged cells. Cells that were negative for both markers were characterized
as viable. Approximately 50,000 events were measured for each sample. Due to sample
availability, only one time point (24 h) was measured on one cell line (SNU449) at
two concentrations (10 and 100 μg/ml). As such, these data have been placed in the
Additional file
1.

Real-time optical bright-field microscopy

Hep3B cells were cultured in glass bottom (no. 1.5) 24-well plates purchased from
MatTek Corporation (Ashland, MA, USA). After overnight incubation, the cells formed
non-confluent monolayers. The 24-well plate was placed in an incubator enclosing a
1X81 Olympus microscope (Center Valley, PA, USA) equipped with a DSU Confocal Attachment
and a ×60 oil immersion objective. The cells were allowed to equilibrate with the
incubator environment (37°C, 5% CO2) before adding pre-warmed SGSs and acquiring images. Eight Z-plane images were acquired with a gap of 1 μm every 15 min. A typical experiment
comprised of 10 to 15 waypoints. In-focus light from all planes was merged and is
represented in the still shots and the movies. Hep3B cells with no exposure to SGS
were also imaged as a control.

Transmission/scanning electron microscopy

For transmission electron microscopy (TEM) imaging, 25,000 Hep3B or SNU449 cells were
plated in 12-well plates. After 24 h, the cells were exposed to the SGS at 10 μg/ml
for 24 h. The media was removed, and cells were washed twice with PBS. The cells were
then harvested after trypsinization and washed once more with PBS. Finally, the cells
were resuspended in Trump’s Fixative (BBC Biochemical, Seattle, WA, USA). Samples
were washed with 0.1% cacodylate-buffered tannic acid, treated with 1% buffered osmium
tetroxide, and stained with 1% uranyl acetate. The samples were ethanol dehydrated
and embedded in LX-112 medium. After polymerization, the samples were cut with an
UltraCut E Microtome (Leica, IL, USA), double stained with uranyl acetate/lead citrate
in a Leica EM stainer, and imaged with a JEM 1010 TEM (Jeol USA, Inc., Peabody, MA,
USA) at an accelerating voltage of 80 kV. Images were acquired with an AMT Imaging
System (Advanced Microscopy Techniques Corp., Woburn, MA, USA). For SEM, the cells
were prepared in a similar manner. The dried samples were coated with a 35-nm-thick
platinum layer. Samples were imaged using a JSM 5900 scanning electron microscope
(JEOL USA, Inc.) equipped with a backscatter electron detector and digital camera.
The beam energy was 5 kV.

Results and discussion

SGS characterization

As can be seen in Figure
1, AFM statistical analysis showed the majority of SGSs (sample size 61) to be approximately
1.41 ± 0.08 μm in diameter with a height of approximately 1.01 ± 0.02 nm, indicating
mainly individualized SGSs
[22,23]. In some instances, there was also evidence of larger SGSs of diameter approximately
5.5 μm (Additional file
1: Figure S1). Raman spectra of the initial graphite material and an SGS sample are
depicted in Additional file
1: Figure S2. According to previous Raman studies
[4], graphene can be identified by monitoring the position of the 2D band, whereby sulfonation
of the phenyl groups and subsequent formation of the SGS sodium salt lead to repulsive
interactions between the SO3− groups (to produce exfoliation), as evidenced by a slight shift in the 2D peak in
Additional file
1: Figure S2. Functionalization by sulfonation has also been confirmed by XPS and TGA,
which is provided in Additional file
1: Figures S3 and S4, respectively. Taken together, these data characterize the SGS
samples as being made up of both individualized SGSs and stacked SGSs of diameters
ranging from 1.41 to 5.5 μm.

Cytotoxicity profiles of SGSs

MTT assay analysis over 5 days showed both a time- and concentration-dependent cytotoxicity
profile for both cell lines. Control samples were also used in conjunction with the
in vitro samples to take into account an increase in 570-nm photon absorption due to the SGSs
themselves, which could obscure correct interpretation of the results. As can be seen
in Figure
2A, although the SNU449 and Hep3B cell lines were approximately 80% to 90% viable after
24 h upon exposure to SGS concentrations of 0.1 to 10 μg/ml, the highest concentration
of 100 μg/ml resulted in a drastic drop in viability to 60% and 20% for SNU449 and
Hep3B cells, respectively. This decrease in viability occurred over time until almost
complete necrosis of cells at 72 h. For lower concentrations, while the Hep3B cells
seem to tolerate SGS better, the SNU449 cells had the greater viability (approximately
50%) for the 10 μg/ml concentration after a 5-day period. The WST-1 results shown
in Figure
2B depict both a weak concentration- and time-dependent cytotoxicity profile. The viability
of Hep3B cells generally stays within the 90% range and only decreases to approximately
70% for the highest concentration. This is also similar for the SNU449 cells which
show a constant viability of approximately 90% to 135% for concentrations 0.1 to 10
μg/ml and a loss in viability down to 80% after a period of 48 to 72 h for the maximum
concentration of 100 μg/ml. Finally, the release of intracellular LDH can provide
evidence of plasma membrane damage. Figure
2C shows minimal membrane damage as evidenced by minimal LDH release in both cell lines
after 72 h of exposure to SGS for concentrations up to 100 μg/ml.

Figure 2.Cytotoxicity Data (MTT, WST-1, and LDH). MTT (A), WST-1 (B), and LDH (C) assays of SNU449 and Hep3B cancer cell lines. As a function of time and SGS concentration.

Previous work by Zhang et al.
[18] demonstrated a similar MTT concentration-dependent viability profile with neural
phaeochromocytoma-derived PC12 cells exposed to graphene synthesized via CVD (purified
using a diluted hydrochloric acid wash with sonication). They showed cell viability
of approximately 40% after 24 h of exposure to their graphene particles at a concentration
of 100 μg/ml, which is similar to MTT values seen in this work. In comparison, Chang
et al. also demonstrated a concentration-dependent profile which was however not time
dependent since they observed similar viability profiles at 24, 48, and 72 h
[16].

Although the MTT and WST-1 profiles are generally identical for time periods 24 to
72 h (with possibly the exception of the WST-1 results which show a weak time-dependent
and concentration-dependent response), the major difference is the drastic loss in
viability for concentrations of 100 μg/ml observed in the MTT assay. This observation
could be explained by interactions of SGSs with insoluble MTT formazan crystals (formed
after the enzymatic reduction of MTT within the cells) which stabilize their structure
and prevent them from becoming solubilized by DMSO. This has already been observed
by Wörle-Knirsch et al.
[24]. In their work, they showed that single-walled carbon nanotubes (SWNTs) were found
to be non-toxic when using assays such as LDH, annexin V, and PI staining, mitochondrial
membrane potential, as well as other tetrazolium salt-based water-soluble assays such
as WST-1, XTT, or INT. However, the MTT assay was the only assay which displayed SWNT
cytotoxicity.

In addition, real-time bright-field microscopy (Figure
3) did not show any morphological features suggestive of cytotoxicity, such as blebbing,
membrane rupture, pyknosis, or fragmentation, for concentrations 1 to 10 μg/ml. Also,
several cells were observed undergoing mitosis (data not shown). These findings suggest
that at these low concentrations, the sulfonation process affords protection to cells
against the cytotoxic effects of graphene, similar to the observed protein corona-mediated
mitigation of GO cytotoxicity recently published by Hu et al.
[17]. However, there was a drastic change in cell morphology for concentrations of 100
μg/ml which shows evidence of pyknosis and fragmented, spindle-like cell features
for the SNU449 cell lines. In these regard, we suggest that 10 μg/ml should be the
upper concentration limit when using SGSs for full biocompatibility and that more
work should be undertaken to understand the exact death mechanism of SGSs at concentrations
>10 μg/ml. We initially sought to investigate this through the use of propidium iodide
and annexin V FITC staining with cell flow cytometry, but as mentioned in the ‘Methods’
section, we could only perform one time slot (24 h) with one cell line (SNU449) at
two concentrations (10 and 100 μg/ml).

Propidium iodide is a cell impermeable fluorophore that can bind to the DNA of cells
which have lost nuclear and plasma membrane integrity. From our fluorescence-activated
cell sorting (FACS) analysis shown in Additional file
1: Figure S5, we found that with an increasing concentration of SGS nanoparticles,
the intensity of positive PI-stained cells increased from approximately 1.9% to 10.3%,
suggesting slight cell membrane structural damage, while the majority of cells remain
healthy and viable at approximately 93% ± 2.4%. Phosphatidylserine (PS) externalization
is an early event in the apoptosis cascade. Annexin V binds to PS with high affinity.
Our FACS analysis hence also demonstrates that very few cells were annexin V positive
24 h after exposure to SGS which ruled out apoptosis as a significant cell death mechanism,
as was similarly reported for GO materials
[16,18].

Cellular internalization of SGSs

Figure
4 depicts high-resolution SEM images of both SNU449 and Hep3B cancer cells after exposure
to SGS at a concentration of 10 μg/ml for 24 h. Control samples (no SGSs) are shown
in the Additional file
1: Figure S6. All samples were first coated with a 35-nm layer of platinum before imaging.
The cells were approximately 10 to 25 μm in diameter and heterogeneous in nature.
Figure
4A showed what is likely to be variability in surface coating of the platinum layer.
When comparing the left and right images of the SNU449 cellular structures in Figure
4A, the left side has what looks like a thicker layer of platinum, which seems to be
filling more of the space between adjacent pseudopodia structures. Comparing Figure
4A and Figure
4B, it can clearly be seen that a relatively large structure is protruding out of a
SNU449 cell in two locations. These structures appear to be graphite (i.e., multiple
stacked SGS) of thickness approximately 500 nm which the cell has internalized. Figure
4C depicts another large nanoplatelet of stacked SGS, which is effectively compressing
a Hep3B cell and deforming the cellular structure. Figure
4D and Figure
4E are the most interesting figures since they display evidence of cellular internalization,
folding, and compartmentalization of SGS.

In Figure
4D, it appears as if the Hep3B cell is actively internalizing multiple, stacked SGS
of height approximately 35 nm, but is most likely a single SGS which looks thicker
due to the platinum layer. The folding phenomenon is also evident in Figure
4E where folding of SGS can be seen in the bottom left corner and bottom midsection
of the image, as indicated by the white arrows. There is also evidence of slightly
deformed SGS on top of the cellular surface in the upper right-hand section. Finally,
Figure
4F depicts the images of both SGS deformation and internalization of large pieces of
graphitic materials. The appearance of pseudopodia over the surface of the SGS is
indicated by the red arrows.

Cellular internalization of the SGS using microtome high-resolution TEM was then investigated,
as shown in Figure
5. Uranyl acetate was used as a negative staining agent. Although single-sheet graphene
should appear close to transparent in TEM imaging, we believe visualization of the
SGS in the TEM images is due to uranyl ions binding to the functionalized graphene
sheets (which would result in a darker image) or that they are stacked graphene layers
which are reducing the optical transparency. From the outset, we suspected that there
was some cellular internalization of submicron-sized amorphous carbonaceous materials
present in the initial graphite material from which the SGS were obtained. Evidence
of this can be found in the Additional file
1: Figure S1. Furthermore, Figure
5A,C indicates cellular internalization of these materials since there is no evidence
of structural uniformity or stacking, which can usually be seen for graphene by TEM
at this resolution
[25]. However, Figure
5B clearly shows compartmentalization of SGS, and closer examination reveals a network
of lines (red arrows) throughout this structure, which look exactly like the folded
graphene sheets previously reported by A. K. Geim et al.
[25]. A magnified view of this key figure is shown in Additional file
1: Figure S7.

Figure 5.SGS Internalization within Hep3B cancer cells. TEM images of internalized carbonaceous material and SGSs within Hep3B liver cancer
cells (A to F). Figure
4D,E,F is of the same cell

Figure
5D,F shows close up images of two areas of Figure
5E to reveal a stained black circular particle (Figure
5E) and a more transparent, slightly smaller, circular particle (Figure
5F). As these particles are of the same diameter as the SGS previously characterized,
they are likely SGS that have internalized into the cell without folding or compartmentalization.
As previously indicated, the large difference in contrast between these two SGS structures
could be due to uranyl ions binding to the functionalized SGS or due to multiple stacked
graphene layers.

It should be noted that the cellular internalization of large SGS caused artifacts
in some instances during the microtome procedure. This can be seen in Figure
6 where there is a large area of internalized SGS adjacent to a completely transparent
‘hole’. This hole is most likely caused by the microtome blade contacting the SGS
and removing the structure from the cellular cytoskeleton (thus leaving behind an
SGS footprint). There is also some evidence of this in Figure
5A where the carbonaceous NP seems to have been dislodged from its initial position,
leaving behind a transparent hole in the left image. This result also serves as good
evidence of the cells’ ability to internalize relatively large pieces of graphite
yet still remain healthy.

Figure 6.TEM image of microtome cutting artifacts caused by SGS inside a SNU449 cell. It is likely that some large chunks of graphite and/or SGS have been dislodged from
the transparent region in the top right corner of the image.

Using real-time bright-field optical microscopy, we could also track the internalization
of SGSs in liver Hep3B cells as a function of time (over a 17-h period). As can be
seen in Figure
7, when looking at snap shots from approximately 10 to 17 h, there were two large SGS
(indicated by red and blue arrows) which became attached to the cell membrane and
gradually internalized into the cell - as is evidenced by the loss of resolution and
blurred nature of the SGS images. Furthermore, the cell retracted to undergo mitosis
once the trapped particles are internalized. (Figure
7E,F,G,H, full movie also available in the Additional file
2: Hep3B SGS movie and Additional file
3: Hep3B control movie).

Figure 7.Optical bright-field images of SGS internalization within Hep3B cancer cells across a 17-h period. Two SGS particles of diameters approximately 2 μm, indicated by red and blue arrows,
are shown to be internalizing approximately 10 h after exposure to SGS of concentration
10 μg/ml. The two cells visible seem to be undergoing cell division. (A to H) Time points at 10 to 17 h, in 1-h increments.

Given that graphene is thought to be the hardest material known
[3], it is counterintuitive to believe that liver carcinoma cells are capable of folding
and compartmentalizing graphene sheets. However, if these sheets contained structural
defects such as point defects, single vacancies, multiple vacancies, carbon adatoms,
dislocation-like defects, or edge defects, as extensively reviewed by Banhart et al.
[26], the cells may be able to fold the sheets, one at a time, along these defect lines
(in a ‘shedding nature’) and compartmentalize them within phagosomes or vesicles using
reasonably low-energy processes. The defect content of the SGS, in relation to the
starting graphite material, can be indicated by the relative intensity of the Raman
D band to G band ratio, located at approximately 1,350 and 1,580 cm−1, respectively
[27]. Although the synthesis procedure and Raman characterization shown in Additional
file
1: Figure S2 shows a weak D band enhancement after exfoliation due to functionalization
of the graphitic edges, it remains unclear as to what defects, if any, are inherent
to the graphene nanoplatelets.

Conclusions

We have investigated the cytotoxicity and internalization of highly exfoliated, water-soluble
SGSs when exposed in vitro to highly aggressive human liver cancer cells (SNU449 and Hep3B). Both MTT and WST-1
colorimetric assays displayed a similar concentration- and time-dependent cytotoxicity
profile for concentrations of 0.1 to 10 μg/ml. These trends were also evident from
LDH observations. However, the SGSs seemed to be toxic to both cell lines at the highest
concentration of 100 μg/ml. We have also observed an interesting cellular internalization
phenomenon for graphene materials for the first time. The cancer cells were capable
of internalizing relatively large SGSs with diameters comparable to the cells themselves
as well as smaller SGS having heights indicative of single graphene sheets. Although
not conclusive, there is evidence to suggest that due to graphene structural defects,
the cancer cells are also able to actively fold and compartmentalize these sheets.
We speculate that the findings reported here may encourage the development of SGSs
for applications in drug delivery, medical imaging, and even hyperthermic cancer therapy
by NIR and/or radio frequency heating. To date, such applications have been explored
for more rigid carbon nanostructures such as fullerenes
[28] and nanotubes
[29-32], but a non-toxic, more flexible (foldable), and larger surface-area material as provided
by graphene offers an alternative design strategy.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SJC conceived the study, interpreted the results, guided the contributing authors
in their research, performed the optical bright-field imaging (alongside MR), and
wrote the manuscript. MR performed the MTT assay study, helped with the TEM/SEM imaging,
and worked with SJC on the optical bright-field imaging studies. BTC carried out the
LDH assay. OK synthesized and supplied the SGSs. KM and WDK performed FACS on the
SNU449 cell line. MAC performed the AFM imaging of the SGSs. WEB, LJW, and SAC participated
in the design of the experiments, acted as mentors for the authors, and extensively
reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgments

This work was funded by the NIH (U54CA143837), the NIH M.D. Anderson Cancer Center
Support Grants (CA016672), the V Foundation (SAC), The Welch Foundation (C-0627, LJW;
C-0490, WEB), and an unrestricted research grant from the Kanzius Research Foundation
(SAC, Erie, PA, USA). We thank Kristine Ash from the Department of Surgical Oncology,
M.D. Anderson Cancer Center for the administrative assistance, Kenneth Dunner, Jr.
of The High Resolution Electron Microscopy Facility at The University of Texas M.D.
Anderson Cancer Center (NCI Core grant CA16672) for providing TEM imaging services,
and Jared Burks of the Cytometry and Cellular Imaging Core Facility (NIH MDACC support
grant CA016672) for providing invaluable assistance with real-time optical imaging.